Human Thromboxane A2 Receptor Gene Research Articles

Transcriptional regulation of the human thromboxane A2 receptor gene by Wilms' tumor (WT)1 and hypermethylated in cancer (HIC) 1 in prostate and breast cancers

The prostanoid thromboxane (TX) A2 plays a central role in hemostasis and is increasingly implicated in neoplastic disease, including prostate and breast cancers. In humans, TXA2 signals through the TPα and TPβ isoforms of the T prostanoid receptor, two structurally related receptors transcriptionally regulated by distinct promoters, Prm1 and Prm3, respectively, within the TP gene. Focusing on TPα, the current study investigated its expression and transcriptional regulation through Prm1 in prostate and breast cancers.Expression of TPα correlated with increasing prostate and breast tissue tumor grade while the TXA2 mimetic U46619 promoted both proliferation and migration of the respective prostate (PC3) and breast (MCF-7 and MDA-MD-231) derived-carcinoma cell lines. Through 5′ deletional and genetic reporter analyses, several functional upstream repressor regions (URRs) were identified within Prm1 in PC3, MCF-7 and MDA-MB-231 cells while site-directed mutagenesis identified the tumor suppressors Wilms' tumor (WT)1 and hypermethylated in cancer (HIC) 1 as the trans-acting factors regulating those repressor regions. Chromatin immunoprecipitation (ChIP) studies confirmed that WT1 binds in vivo to multiple GC-enriched WT1 cis-elements within the URRs of Prm1 in PC3, MCF-7 and MDA-MB-231 cells. Furthermore, ChIP analyses established that HIC1 binds in vivo to the HIC1(b)cis-element within Prm1 in PC3 and MCF-7 cells but not in the MDA-MB-231 carcinoma line. Collectively, these data establish that WT1 and HIC1, both tumor suppressors implicated in prostate and breast cancers, transcriptionally repress TPα expression and thereby provide a strong genetic basis for understanding the role of TXA2 in the progression of certain human cancers.

Regulation of the human thromboxane A2 receptor gene by Sp1, Egr1, NF-E2, GATA-1, and Ets-1 in megakaryocytes

The alpha and beta isoforms of the human thromboxane A(2) (TXA(2)) receptor (TP) are encoded by a single gene but are transcriptionally regulated by distinct promoters, termed promoter 1 (Prm1) and Prm3, respectively. Herein, it was sought to identify factors regulating Prm1 within the megakaryocytic human erythroleukemia 92.1.7 cell line. Through gene deletion and reporter assays, the core Prm1 was localized to between nucleotides -6,320 and -5,895, proximal to the transcription initiation site. Furthermore, two upstream repressor and two upstream activator regions were identified. Site-directed mutagenesis of four overlapping Sp1/Egr1 elements and an NF-E2/AP1 element within the proximal region substantially reduced Prm1 activity. Deletion/mutation of GATA and Ets elements disrupted the upstream activator sequence located between -7,962 and -7,717, significantly impairing Prm1 activity. Electrophoretic mobility shift assays and chromatin immunoprecipitations confirmed that Sp1, Egr1, and NF-E2 bind to elements within the core promoter, whereas GATA-1 and Ets-1 factors bind to the upstream activator sequence (between -7,962 and -7,717). Collectively, these data establish that Sp1, Egr1, and NF-E2 regulate core Prm1 activity in the megakaryocytic-platelet progenitor cells, whereas GATA-1 and Ets-1 act as critical upstream activators, hence providing the first genetic basis for the expression of the human TXA(2) receptor (TP) within the vasculature.

Characterization and chromosomal mapping of the human thromboxane A2 receptor gene.

The human thromboxane A2 gene, present as a single copy, spans over 15 kilobases (kb) and contains 3 exons divided by 2 introns. The first intron, intron 1, exists in the 5'-noncoding region, 83 base pairs upstream from the ATG start site and is 6.3 kb long. Intron 2 with a length of 4.3 kb is located at the end of the sixth transmembrane region, thereby separating it from the downstream coding sequences including the seventh transmembrane region and the 3'-untranslated region. By rapid amplification of 5'-cDNA ends, transcription initiation sites starting in two different putative promoter regions were determined. In the 5'-flanking region of these transcription initiation sites, no typical TATA box exists. The major promoter, the promoter region I, contains four potential SP-1 binding sites and several potential binding sites for other transcription factors. By polymerase chain reaction analysis, a small portion of the RNA transcribed from this gene was shown to contain an additional 115-base pair long noncoding exon, exon 1b, which is located in intron 1. No additional exon in intron 2 was detectable, indicating that a single type of thromboxane A2 receptor protein is encoded by this gene. Chromosomal localization was carried out by fluorescence in situ hybridization of cloned genomic DNA to the metaphase chromosome. The gene was assigned to 19p13.3 of human chromosome.